Antimicrobial resistance (AMR) is a One-Health (human, animal, environment) challenge that requires collaborative, interdisciplinary action. Comparable surveillance data are needed to effectively inform policy interventions aimed at preventing the spread of AMR. Environmental monitoring lags behind that of other One Health sectors and is in need of agreed upon targets and standardized methods. A challenge is that there are numerous microorganisms, antibiotic resistance genes (ARGs), and mobile genetic elements and corresponding methods that have been proposed. In this dissertation, a framework for AMR monitoring of aquatic environments was developed through a combination of literature review and stakeholder input, via surveys and a workshop. Through this process, three targets were selected for standardization: the sulfonamide resistance gene (sul1), the class 1 integron integrase gene (intI1), and cefotaxime-resistant Escherichia coli. Quantitative polymerase chain reaction (qPCR)- and culture-based protocols were developed and pilot tested in two independent laboratories on a set of six water matrices: wastewater, recycled water, and surface water from six different wastewater utilities engaging in water reuse located in five states across the USA. The impact of wastewater treatment and advanced water treatment processes was examined in terms of removal of these targets. Finally, qPCR and culture methods were used to examine the relationship between sul1, intI1, E. coli, and fecal indicators in private household wells across four states in the Southern USA that were identified as susceptible to storm events. The overall findings provide a useful baseline occurrence of the proposed AMR monitoring indicators across a range of water types and protocols that are accessible to water utilities. / Doctor of Philosophy / Life-saving drugs and treatments are failing at an increasing rate because of antimicrobial resistance (AMR). Antimicrobials, such as antibiotics, are a double-edged sword, because they are an effective weapon for killing disease-causing pathogens, but the more they are used the greater the likelihood that microbes that are resistant to them will survive, reproduce, and spread. National action plans for AMR have been created by a majority of countries, emphasizing the importance of antibiotic stewardship and other mitigation strategies. However, numerous data gaps need to be addressed in order to identify strategies that are most likely to be effective and to implement them. Environmental surveillance, including wastewater influent, wastewater effluent, and surface water, could prove an informative means to track AMR trends with time and relate them to human activities and corresponding mitigation efforts. The purpose of this dissertation was to develop a framework for AMR surveillance of aquatic environments and to test it across an array of sample types. We considered an array of possible culture- and DNA-based targets from available scientific literature and engaged experts and stakeholders in narrowing down the list to options that were both informative and feasible. We developed protocols for quantifying an antibiotic resistance gene (sul1), a mobile genetic element that has been implicated in the spread of multi-antibiotic resistance (intI1), and an extended spectrum beta-lactamase (ESBL) producing form of Escherichia coli. We compared the methods between two independent laboratories on untreated wastewater, treated wastewater, recycled water, and surface water collected from six locations across five states. We additionally did a survey of private household well water that was hypothesized to be vulnerable to contamination due to storms and lack of resources for maintenance. The results of this research can help to support environmental monitoring of AMR across the US and globally.

Widespread use of antibiotics in livestock production can result in the dissemination of bacteria carrying antibiotic resistance genes (ARGs) to the broader environment. Within agroecosystems, ARGs can pose a risk to livestock handlers, farmers, and ultimately consumers. The overall goals of this dissertation are to examine the presence of resistance (antibiotic, metal) in agricultural soils and evaluate the most critical potential points of best management control of antibiotic resistance spread along the agricultural production chain. The relative impacts of agricultural practices, manure management, native soil microbiota, and type of crop grown and harvested on the agricultural resistome are multi-dimensional and cannot be captured via a single analytical technique or by focusing on one specific point in the agricultural process. Culture-, molecular "indicator"-, and next-generation sequencing- based methods were employed to characterize antibiotic resistance via taxonomic and functional profiles on the broader manure, soil, and vegetable surface microbial communities through 16S rRNA amplicon sequencing and shotgun metagenomics. Although antibiotic concentrations dissipated in the soil after 28 days after amendment application, antibiotic resistance presence was recoverable throughout the entire 120d growing season in the compost and manure amendments, the amended soil, and on vegetable surfaces. The addition of organic fertilizers increased antibiotic resistance presence compared to background levels. Further, metals and metal resistance were also measured in the amended soils and were found to be at greater levels in the inorganically fertilized soils compared to the manures and compost amended soils. Analysis of the widespread agroecosystem microbial community composition and broader metagenome has characterized varying genera profiles in the soil and on the vegetable surfaces and specific ARG and mobile genetic element (plasmid) co-occurrences. These co-occurrences highlight which ARGs may be most critical for future antibiotic resistance dissemination research. It is imperative to employ multiple methods when measuring agricultural resistance, as one method alone may miss significant patterns and lead to different best management recommendations. Linking the livestock manure, soil, and vegetable surface-associated ARBs, ARGs, resistomes, and microbiomes will help identify critical control points for mitigation of agricultural dissemination of antibiotic resistance to the environment and food production. / Doctor of Philosophy / By 2050, it is estimated that antibiotic resistant infections will be the leading cause of death worldwide. It is important to consider human, animal, and environmental health when researching antibiotic resistance, which is known as a "One Health" approach. In this dissertation work, I focus on the environmental side of antibiotic resistance in our agricultural systems. Agriculture is a known source of antibiotic resistance due to its use of antibiotics in livestock as a treatment for illness, and in some instances, as a growth promoter. Over one growing season, I measured antibiotic resistance in an agricultural setting using many techniques. First, I analyzed the effects of inorganic (chemical) versus organic (manure and compost) fertilization on antibiotic resistance in the soil. I measured antibiotic resistance by growing antibiotic resistant bacteria, quantifying specific antibiotic resistant genes (ARGs) using DNA amplification, and quantifying all the ARGs in the soil using a next-generation sequencing (NGS) technique called shotgun metagenomics. I found that adding manure to the soil increases ARGs compared to background soil levels, and that composting in an effective management strategy in decreasing ARGs in the soil over time. Second, I analyzed the same effects of fertilization on metal resistance in the soil. I was able to use the same NGS dataset to measure metal resistance genes (MRGs). I found that adding inorganic chemical fertilizer increases MRGs in the agricultural soils compared to the organic (manure or compost) fertilizer. Additionally, I studied the microbes that live in the agricultural soils using another kind of NGS data specific for microbial identification. I found that although there were small differences between the microbial populations in the soil when fertilizers were added, they returned to similar composition over the growing season. Lastly, I measured antibiotic resistance and microbes throughout the entire agricultural system. I picked the point of fertilization (manure management), soil, and the lettuce surface to evaluate if antibiotic resistance spreads from the farm to the vegetable that ends up on a consumer's plate. I found that at each point antibiotic resistance is present, but at different levels. Composting reduces ARGs compared to raw manure. Agricultural soils may act as a natural buffer to antibiotic resistance. Lettuce plants grown in compost fertilized soils have less ARGs than lettuce plants grown in manure. There are many agricultural management practices that effectively reduce antibiotic resistance and using all of them plus many measurement methods will ultimately help farmers and consumers reduce antibiotic resistance in our agricultural systems.

Landfilling is the most widely used method for the disposal of municipal solid wastes (MSW) in the United States due to its simplicity and low cost. According to the 2014 report on Advancing Sustainable Materials Management by the USEPA, only 34% of the total MSW generated in the US was recycled, while 13% was combusted for energy recovery. In 2014, 53% of the MSW generated, (i.e. 136 million tons) in the US was landfilled. The treatment of landfill leachates, generated by percolation of water through the landfill, primarily due to precipitation, has been found to be one of the major challenges associated with landfill operation and management. Currently, leachates from most landfills are discharged into wastewater treatment plants, where they get treated along with domestic sewage. Issues associated with treatment of landfill leachates due to their high nitrogen and heavy metal content have been widely studied. Recently, it has been observed that the organic carbon in landfill leachates, specifically humic and fulvic acids (together referred to as "humic substances") contain aromatic groups that can absorb large amounts of ultraviolet (UV) light, greatly reducing the UV transmissivity in wastewater plants using UV disinfection as the final treatment step. This interference with UV disinfection is observed even when landfill leachates constitute a very small fraction (of the order of 1%) of the total volumetric flow into wastewater treatment plants. Humic substances are present as dissolved organic matter (DOM) and typically show very low biodegradability. Removing these substances using chemical treatment or membrane processes is an expensive proposition. However, the concentrations of humic substances are found to be reduced in leachates from landfill cells that have aged for several years, suggesting that these substances may be degraded under the conditions of long-term landfilling. The primary objective of this research was to use a two-stage process employing thermophilic pretreatment followed by a mesophilic anaerobic membrane bioreactor (AnMBR) to mimic the conditions of long-term landfilling. The AnMBR was designed to keep biomass inside the reactor and accelerate degradation of biologically recalcitrant organic carbon such as humic substances. The treatment goal was to reduce UV absorbance in raw landfill leachates, potentially providing landfills with an innovative on-site biological treatment option prior to discharging leachates into wastewater treatment plants. The system was operated over 14 months, during which time over 50% of UV-quenching organic carbon and 45% of UV absorbance was consistently removed. To the best of our knowledge, these removal values are higher than any reported using biological treatment in the literature. Comparative studies were also performed to evaluate the performance of this system in treating young leachates versus aged leachates. Next-generation DNA sequencing and quantitative PCR (qPCR) were used to characterize the microbial community in raw landfill leachates and the bioreactors treating landfill leachate. Analysis of microbial community structure and function revealed the presence of known degraders of humic substances in raw as well as treated landfill leachates. The total number of organisms in the bioreactors were found to be higher than in raw leachate. Gene markers corresponding to pathogenic bacteria and a variety of antibiotic resistance genes (ARGs) were detected in raw landfill leachates and the also in the reactors treating leachate, which makes it necessary to compare these ARG levels with wastewater treatment in order to determine if leachates can act as sources of ARG addition into wastewater treatment plants. In addition, the high UV absorbance of leachates could hinder the removal of ARBs and ARGs by UV disinfection, allowing their release into surface water bodies and aiding their proliferation in natural and engineered systems. / Ph. D.

Antibiotics are biologically active and are globally used in humans and animal medicine for treatment and in sub-therapeutic amounts as growth promoters in animal husbandry, aquaculture and agriculture. After excretion, inappropriate disposal and discharge from drug production facilities they enter into water bodies either as intact drugs, metabolites or transformed products. In water environments they promote development of antibiotic resistance genes (ARGs) which could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. Measurement of antibiotics has been revolutionized with the usage of solid phase extraction (SPE) for enrichment followed by Liquid chromatography mass spectrometry (LC-MS). On-line SPE coupled to LC-MS/MS has the advantages of high sample throughput, low sample preparation time and minimal solvent utilization.  Constructed wetlands (CWs) are potential alternatives to conventional treatment plants to remove organic pollutants. A study at Plönninge, Halmstad was performed to assess the impact of bacterial community pattern and development of resistance in spiked (n=4) and control (n=4). CWs were spiked with antibiotics at environmentally relevant concentrations continuously for 25 days. Shannon Index (H’) were used to determine the bacterial diversity and real-time PCR detected and quantified antibiotic resistance genes (ARGs) sulI, tetA, tetB, erm, dfrA1, qnrS and vanB and class 1 integrons intI1. No significant differences in bacterial compositions or in ARGs or integron concentrations could be discerned between exposed and control wetlands. A study conducted in Northern Pakistan showed that the antibiotic levels in most studied rivers were comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics were detected in the river in close vicinity of the 10 million city Lahore, e.g. 4600 ng L−1 sulfamethoxazole. Highest detected levels were at one of the drug formulation facilities, with measured levels up to 49000 ng L−1 of sulfamethoxazole for example. The highest levels of ARGs detected, sul1 and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. In the study in UK, sewage epidemiology surveillance is used to measure the oseltamivir carboxylate (OC), metabolite of oseltamivir (parent drug) in twenty four time proportional hourly influent samples from two WWTPs and then back-calculations were made to assess the compliance of drug.  Predicted users of oseltamivir, based on measured OC in waste water, ranged from 3-4 and 120-154 people for the two WWTP catchments, respectively, which are consistent with the projected use from national antiviral allocation statistics, 3-8 and 108-270, respectively. Scenario analysis suggests compliance was likely between 45-60% in the study regions.